A purple acid phosphatase gene associated with cotton lint content and its application

Abstract

The present invention provides a purple acid phosphatase gene associated with cotton lint percentage and its application, belonging to the field of biotechnology application. The cDNA sequence of the gene GhPAP18 in tetraploid upland cotton TM-1 is: SEQ ID NO.1, the sequence of the encoded amino acid is: SEQ ID NO.2, and the genomic DNA sequence is: SEQ ID NO.3. The GhPAP18 gene is regulated by an adjacent SNP site (D13:50288965), and the SNP base mutates from cytosine (C) to thymine (T), and the genotype lint percentage after mutation is significantly higher than that before mutation. The present invention also provides a method for identifying high lint percentage cotton varieties and an application of cultivating new cotton varieties with high lint percentage by genetic engineering means.

Description

Technical Field

[0001] The present invention relates to genes associated with cotton lint content and applications, in particular to purple acid phosphatase genes associated with cotton lint content and applications, belonging to the field of biotechnology applications. Background Art

[0002] Cotton is one of the most important economic crops in the world, and the development of the cotton industry is related to the national economy and people's livelihood. The yield of cotton fiber has become an important factor restricting the development of the cotton industry. Therefore, it is urgent to cultivate high-yield and high-quality cotton varieties. By exploring the core regulatory genes related to fiber development and analyzing the regulatory network of cotton fiber development, a theoretical basis can be provided for cotton breeding.

[0003] Fiber yield traits are mostly quantitative traits controlled by multiple genes. Genome-Wide Association Study (GWAS) is one of the powerful means to analyze complex plant traits. Existing studies have shown that GWAS can accurately identify key genes related to fiber yield in cotton. Huang et al. resequenced 243 Asian cotton and grass cotton materials and linked the gene GaSTF9, which is significantly associated with Verticillium wilt resistance, through GWAS analysis. The study found that inhibiting the expression of this gene can make the disease-resistant varieties appear susceptible to the disease. This shows that GaSTF9 plays a regulatory role in the process of cotton resistance to Verticillium wilt (Du X, Huang G, He S, et al. Resequencing of 243 diploid cotton accessions based on an updated A genome identifies the genetic basis of key agronomic traits[J]. Nature Genetics, 2018, 50(6):796-802.). Hua Jinping and Kong Jie's team resequenced a population of 336 self-bred and introduced varieties of sea island cotton, and conducted GWAS analysis based on multiple traits such as fiber yield and quality. They found that the gene GbLP1 existed on A05 and was highly expressed in the early stages of fiber development in high lint percentage materials, suggesting that this gene may be involved in fiber yield formation (Zhao N, Wang W, Grover CE, et al. Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fibre length, lint percentage and Fusarium wiltresistance[J]. Plant Biotechnology Journal, 2022, 20(4):691-710.).GWAS analysis of the sequencing data of 276 upland cotton varieties located the genes Gh_D09G2376 and GD_D06G1908 related to fiber length and strength traits. RT-qPCR verification showed that these two genes were dominantly expressed at multiple stages of cotton fiber development, suggesting that these two genes play a regulatory role in the fiber development process (Liu W, Song C, Ren Z, et al. Genome-wide association study reveals the genetic basis of fiber quality traits inupland cotton (Gossypium hirsutum L.)[J]. BMC Plant Biology, 2020, 20(1):395.). In summary, GWAS analysis can efficiently and accurately screen target genes and accelerate the process of precision breeding.

[0004] Phosphorus is one of the six essential nutrients for plants and is crucial to their growth and development (Ming Wei, Xinwei Wang, Bo Chen, et al. Research progress into the function of purpleacid phosphatase gene family in plants[J]. Chinese Bulletin of Botany, 2019, 54(1): 93-101). The lack of phosphorus can lead to short plants, slow growth, reduced branches or tillers, and incomplete seeds, which seriously affects plant yield. Purple acid phosphatases (PAPs) are acidic nucleases in the binuclear metallodehydrogenase family. They can release phosphorus by catalyzing the hydrolysis of phosphate monoesters, thereby improving the utilization rate of phosphorus in plants (Olczak M, Morawiecka B, Watorek W. Plant purple acidphosphatases - genes, structures and biological function[J]. Acta BiochimPol. 2003, 50(4):1245-1256). Further research on the biological function and molecular mechanism of purple acid enzyme genes in plants is of great significance for analyzing the growth and development process of plants and improving crop yields. In recent years, more and more plant PAPs genes have been identified and studied. Overexpression of AtPAPs under phosphorus deficiency conditions revealed that the length density of the main roots and lateral roots of Arabidopsis plants increased (Wang L, Li Z, Qian W, et al. The Arabidopsis purple acidphosphatase AtPAP10 is predominantly associated with the root surface andplays an important role in plant tolerance to phosphate limitation[J]. Plant Physiology, 2011, 157(1): 1283-1299).In soybean, overexpression of PAPs can cause premature senescence of nodules, affect nitrogen fixation, and thus reduce soybean yield (Wang J, Si Z, Li F, et al. A purple acidphosphatase plays a role in nodule formation and nitrogen fixation inAstragalus sinicus[J]. Plant Molecular Biology, 2015, 88:515-529). Tian et al. found that overexpression of OsPAP10a and OsPAP21b can cause premature flowering in rice (Tian J, Wang C, Zhang Q, et al. Overexpression of OsPAP10a, A root-associated acid phosphatase, increased extracellular organic phosphorus utilization in rice[J]. Journal of Integrative Plant Biology, 2012, 54(1): 631-639). However, in cotton, there are few studies on PAPs, and the molecular mechanism of the activity of PAPs and crop phenotypes is still unclear. Summary of the invention

[0005] In view of the above problems, the present invention provides a gene associated with cotton lint percentage trait and its application.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present invention is: a purple acid phosphatase gene GhPAP18 significantly associated with the cotton lint percentage trait, wherein the purple acid phosphatase gene GhPAP18 associated with the cotton lint percentage has a cDNA sequence as shown in SEQ ID NO:1 in tetraploid upland cotton TM-1; the protein encoded by it has an amino acid sequence as shown in SEQ ID NO.2, and the genomic DNA sequence of the gene is SEQ ID NO.3.

[0007] The SNP marker on the gene containing the above-mentioned gene GhPAP18 sequence is significantly associated with the cotton lint percentage trait, the marker site of the SNP marker is located in the same association analysis interval as the gene GhPAP18 and is located downstream of the gene GhPAP18, and the gene GhPAP18 is regulated by the marker site; the marker site of the SNP marker is the base type of D13:50288965, wherein the cotton with the base T is a high lint percentage cotton variety, and the cotton with the base C is a low lint percentage cotton variety. The base of the SNP site changes from C to T, and the material with the base C of the SNP site in the population has a higher expression of GhPAP18, which is manifested as a lower lint percentage; the material with the base T of the SNP site has a lower expression of GhPAP18, which is manifested as a higher lint percentage.

[0008] The above-mentioned SNP markers are used in the breeding process of new cotton varieties. A gene containing the gene GhPAP18 and the base of the marker site of the SNP marker is T is transferred into a cotton variety to cultivate a new cotton variety with a high lint percentage, or the marker site C on the gene containing the gene GhPAP18 and the base of the marker site of the SNP marker is C is site-specifically mutated to T through genetic engineering to cultivate a new cotton variety with a high lint percentage.

[0009] A primer pair for detecting the marker site of the above-mentioned SNP marker, wherein the upstream primer sequence of the primer pair is shown as SEQ ID NO.7, and the downstream primer sequence is shown as SEQ ID NO.8.

[0010] A kit comprises the above-mentioned primer pair.

[0011] An application of the above-mentioned kit, wherein the kit is used to identify the lint percentage trait of cotton and to screen and cultivate new varieties of cotton with high lint percentage.

[0012] The purple acid phosphatase gene associated with cotton lint content and its application have the following beneficial effects:

[0013] 1. The present invention discovered a purple acid phosphatase gene GhPAP18 associated with cotton lint content through genome-wide association analysis. Figure 1 ), expression level analysis of different tissues and developmental stages of cotton ( Figure 2 ) and regression analysis of relative expression level and lint percentage trait level ( Figure 3 ) are closely related to the characteristics of cotton clothing.

[0014] 2. The breed population can be divided into two categories according to the different genotypes of the SNP (D13:50288965) that regulates GhPAP18. Statistical analysis methods found that there was a significant difference in the lint percentage between the two groups ( Figure 4), further proving the correlation between this gene and cotton yield traits.

[0015] 3. The SNP genotypes of GhPAP18 in relatively high and low lint percentage varieties can be verified by PCR technology, which has the advantages of easy operation, high sensitivity and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0017] Figure 1 This is the result of the association analysis between cotton lint content and the whole genome; in the figure:

[0018] The horizontal axis represents the position on the chromosome (Mb), and the vertical axis represents the significance of the association between the SNP locus and the agronomic trait, expressed as -log 10 (P value) indicates. Arrows indicate SNP variant sites associated with agronomic traits.

[0019] Figure 2 This is a statistical chart of the gene expression level of GhPAP18 in different tissues and developmental stages of cotton; in the figure:

[0020] The horizontal axis represents different tissues, namely root, stem, leaf, ovule, and fiber. The ovule tissue includes 3 days before flowering, 1 day before flowering, the day of flowering, and 1 to 35 days after flowering. The fiber tissue includes 5 days after flowering, 10 days after flowering, 20 days after flowering, and 25 days after flowering. The vertical axis represents FPKM, which is a standard for measuring the relative expression of genes.

[0021] Figure 3 This is the result of regression analysis between the relative expression level of GhPAP18 and the lint content trait level; in the figure:

[0022] Different scattered points represent different variety groups, the horizontal axis represents the standardized gene expression (FPKM), and the vertical axis represents the yield trait lint percentage. The R in the legend in the upper left corner represents the regression coefficient, and p represents the significance level, which is used to evaluate the statistical significance of the fitting curve.

[0023] Figure 4 This is a graph showing the difference in expression and lint percentage between different genotypes of GhPAP18 in a population; in the graph:

[0024] The left figure represents the difference analysis of expression between different genotypes in the population, and the right figure represents the difference analysis of lint percentage between different genotypes in the population. The box plot represents the distribution of lint percentage traits in the variety population. There are 75 and 39 varieties with CC and TT genotypes, respectively. The horizontal line in the box represents the median of the trait distribution. DETAILED DESCRIPTION

[0025] The technical scheme in the embodiment of the present invention is described clearly and completely below in conjunction with the accompanying drawings in the embodiment of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0026] The purple acid phosphatase gene GhPAP18 is significantly associated with the cotton lint content trait. The purple acid phosphatase gene GhPAP18 associated with the cotton lint content has a cDNA sequence as shown in SEQ ID NO:1 in tetraploid upland cotton TM-1; the protein encoded by it has an amino acid sequence as shown in SEQ ID NO.2, and the DNA sequence of the gene is shown in SEQ ID NO.3.

[0027] The gene GhPAP18 described in the present invention is regulated by a downstream SNP (D13:50288965). The base of the SNP site changes from C to T. In the population, the material with the base of the SNP site being C has a higher expression of GhPAP18, which is manifested as a lower lint percentage; the material with the base of the SNP site being T has a lower expression of GhPAP18, which is manifested as a higher lint percentage.

[0028] The application of the gene GhPAP18 of the present invention in identifying high lint percentage upland cotton varieties is specifically: using a specific primer pair for detecting the SNP site D13:50288965, as shown in SEQ ID NO.7 and SEQ ID NO.8, to detect the base type of the SNP site controlling the purple acid phosphatase gene GhPAP18 in the cotton genome, wherein the cotton with the base of thymine (T) is a high lint percentage cotton variety.

[0029] The application of the gene GhPAP18 described in the present invention in breeding new cotton varieties with high lint percentage by genetic engineering means. Specifically, the gene containing the high-yield genotype GhPAP18 (T) can be transferred into cotton varieties by genetic engineering means to improve the lint percentage of cotton, and the SNP site in the low-yield genotype GhPAP18 (C) can also be site-directedly mutated to (T), thereby breeding new cotton varieties with high lint percentage.

[0030] Example 1 Mining of purple acid phosphatase gene GhPAP18 associated with cotton lint content:

[0031] From 2007 to 2009, a detailed survey of cotton yield traits (lint percentage, seed index, etc.) was conducted in Anyang, Henan, Nanjing, Jiangsu, and Kuqa, Xinjiang for 258 modern varieties or strains. At the same time, the whole genome of these 258 cotton varieties was sequenced. The sequencing data was compared with the reference genome of the standard upland cotton line TM-1, and then the software samtools was used to identify SNPs on the basis of the whole genome level, and the mined genetic variation sites and cotton phenotypic data were subjected to whole genome association analysis. The results are as follows Figure 1 As shown, a SNP signal association site (D13:50288965) on chromosome D13 can be associated with the cotton lint percentage trait. At the same time, the SNP is associated with the purple acid phosphatase gene GhPAP18 (GH_D13G1585) in the GWAS signal region. It is preliminarily judged that this gene may be involved in the formation of cotton lint percentage.

[0032] RNA samples from different tissues and developmental stages of the TM-1 upland cotton variety were collected for transcriptome sequencing. Sample materials included roots, stems, leaves, ovules, and fibers. Ovule tissue included 3 and 1 days before flowering, the day of flowering, and 1 to 35 days after flowering. Fiber tissue included 5 to 25 days after flowering. In the results, the GhPAP18 gene was dominantly expressed in ovule tissue 10 days after flowering and fiber tissue 10 days after flowering ( Figure 2 ), that is, it is dominantly expressed in the mid-stage of fiber development, indicating that this gene is related to the constituent factors of cotton lint content traits.

[0033] The relative expression level of GhPAP18 and the level of lint percentage were subjected to regression analysis. The ordinate represents the lint percentage level, and the abscissa represents the FPKM value of GhPAP18 (after normal centering), which represents the relative expression level of GhPAP18. The R in the legend on the upper left corner represents the regression coefficient, and p represents the significance level, which is used to evaluate the statistical significance of the fitting curve. The results of the regression analysis show that the GhPAP18 gene negatively regulates the lint percentage trait ( Figure 3 ).

[0034] The three elements of cotton yield are the number of bolls per plant, the weight of bolls per plant, and the lint percentage. Therefore, by studying the function of this gene in regulating the lint percentage of cotton, it is possible to provide a genetic basis for the improvement of high-yield cotton varieties, thereby promoting the process of precision breeding of cotton. The cDNA sequence of the gene GhPAP18 in tetraploid upland cotton TM-1 is shown in SEQ ID NO.1, the amino acid sequence of the encoded protein is shown in SEQ ID NO.2, and the genomic DNA sequence is shown in SEQ ID NO.3. The cDNA sequence and DNA sequence of GhPAP18 were obtained from the upland cotton genome sequence, and the full-length primers of the gene were designed according to the two ends of the cDNA for PCR amplification. The primer sequences were F1: SEQ ID NO.5 and R1: SEQ ID NO.6. The PCR reaction procedure was as follows: pre-denaturation at 94℃ for 2min; denaturation at 98℃ for 10sec; annealing at 55℃ for 5sec; extension at 68℃ for 10sec, 35 cycles; and finally extension at 68℃ for 5min. The PCR amplification product is sequenced and further compared with cDNA to determine the accuracy of the sequence, and the GhPAP18 gene can be obtained.

[0035] Example 2 Identification of high lint percentage cotton varieties and application in improving yield traits:

[0036] Based on the position of the SNP site (D13:50288965) on chromosome D13 and the sequence nearby (SEQ ID NO.4, the SNP site is at position 51 in the SEQ ID NO.4 sequence), genomic amplification primers were designed at both ends of the SNP site, and the primer sequences were F2: SEQ ID NO.7 and R2: SEQ ID NO.8; this pair of primers was used to perform PCR amplification and sequencing in the DNA of 258 varieties, and the PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds; annealing at 55°C for 5 seconds; extension at 68°C for 10 seconds, 35 cycles; and finally extension at 68°C for 5 minutes. The variety population was divided into two categories according to the different genotypes of the regulatory SNP site: CC represents a low lint percentage genotype, and the SNP site is a homozygous site; TT represents a high lint percentage genotype, and the SNP site is a homozygous mutation. As shown in Tables 1 and Figure 4As shown, the number of varieties with the two genotypes is 75 and 39 respectively (a total of 258 varieties were tested, and there were 4 results classified by genotype, one is the homozygous type, namely the CC, one is the homozygous mutation, namely the TT; another is the heterozygous mutation, the genotype is CT, and the remaining one is the deletion type, that is, the SNP site is missing at this position. In the actual analysis process, the deletion type is generally not considered, and the homozygous mutation is more valuable than the heterozygous mutation. Therefore, the statistical analysis of the present invention only compares the homozygous type CC and the homozygous mutation TT, and the number of varieties with the two genotypes is 75 and 39 respectively). Using the statistical analysis method, it was found that there were significant differences between the homozygous site and the homozygous mutation population, whether between the population genotype and the gene expression level, or between the population genotype and the lint percentage trait level, proving the correlation between the SNP site and the cotton lint percentage trait.

[0037] The above results show that the purple acid phosphatase gene GhPAP18 has important research value in improving lint percentage and breeding new varieties with high lint percentage. On the one hand, molecular markers can be designed based on the two genotypes of the SNP site controlling the purple acid phosphatase gene GhPAP18, which can effectively identify cotton varieties with high lint percentage. On the other hand, the SNP site in the low lint percentage genotype (CC) can be site-directed mutated by genetic engineering to transform it into a high lint percentage genotype (TT) to breed new cotton varieties with high lint percentage.

[0038] Table 1 Identification of high lint percentage and low lint percentage genotypes in population cultivar materials

[0039]

[0040] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. Application of a reagent for detecting SNP markers in the breeding process of new cotton varieties, characterized in that: The SNP marker is used to identify the low lint percentage trait or high lint percentage trait of cotton; The marker site of the SNP marker is located in the same association analysis interval as the gene GhPAP18 and is located downstream of the gene GhPAP18, and the gene GhPAP18 is regulated by the marker site; the marker site of the SNP marker is the nucleotide genotype at position 51 of the sequence shown in SEQ ID NO:4, wherein the cotton with the genotype TT is a high lint percentage cotton variety, and the cotton with the genotype CC is a low lint percentage cotton variety; The gene GhPAP18 is a purple acid phosphatase gene significantly associated with cotton lint content traits, and the genomic DNA sequence of the gene GhPAP18 is shown in SEQ ID NO:

3.

2. The use of a reagent for detecting SNP markers according to claim 1 in the breeding process of new cotton varieties, characterized in that: The upstream primer sequence of the primer pair used to detect the marker site of the SNP marker is shown as SEQ ID NO.7, and the downstream primer sequence is shown as SEQ ID NO.

8.

3. The use of a reagent for detecting SNP markers according to claim 2 in the breeding process of new cotton varieties, characterized in that: The kit for detecting the marker site of the SNP marker comprises the primer pair described in claim 2.

4. An application of a kit for identifying cotton lining properties, characterized in that: The kit is used to identify the lint percentage trait of cotton and screen and cultivate new varieties of cotton with high lint percentage; The kit comprises a primer pair for detecting the marker site of the SNP marker according to claim 1, wherein the upstream primer sequence of the primer pair is shown as SEQ ID NO.7, and the downstream primer sequence is shown as SEQ ID NO.8; The sequence shown in SEQ ID NO:4 was amplified by the above primer pair, and the nucleotide genotype at position 51 of the sequence shown in SEQ ID NO:4 was detected by sequencing. The genotype was CC type, which showed a low lint percentage trait, and was TT type, which showed a high lint percentage trait.

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